Recovery of proteolytic enzymes from pancreas gland residues subsequent to insulin removal



Aug. 10, 1954 PANCREAS RES/DUE AC/D/F/ED WATER STEAM M. THOMPSON RECOVERY OF PROTEOLYTIC ENZYMES FROM PANCREAS GLAND RESIDUES SUBSEQUENT TO INSULIN REMOVAL Filed NOV. 30, 1951 /Z'gl co/voEA/SER COLD MTER RECOVERED ALCOHOL TANK 1N ENTOR.'

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Patented Aug. 10, 1954 RECOVERY OF PROTEIOLYTIC ENZYMES FROM PANCREAS GLAND RESIDUES SUBSEQUENT TO INSULIN REMOVAL William M. Thompson, Chicago, Ill., assignor to Armour and Company,Chicago, Ill., a corporation of Illinois Application November 30, 1951, Serial No. 259,090

This invention relates to the recovery of proteolytic enzymes from pancreas gland residues subsequent to insulin removal. The process of this invention has particular utility in preparing crystalline enzyme products from nog pancreas gland residues.

it has long been known that a number of proteolytic enzymes are obtainable from the pancreas glands of mammals. Commercially, these enzymes are mainly obtained from the pancreas glands of cattle, hogs, and sheep. Pancreas enzyme preparations obtained from these sources have been used in the tanning and textile industries for many years. Recent discoveries indicate that several of the pancreatic enzymes possess therapeutic value. Trypsin has been most extensively investigated for medicinal purposes, but other enzymes such as chymotrypsin, desoxyribonuclease, and ribonuclease appear promising.

The enzymes are present in the pancreas glands in the form oi pro-enzymes or zymogens, which are the precursors of the active enzymes, for example as chymotrypsinogen and trypsinogen. Activation of the enzymes is undesirable, if the insulin is to be extracted from the glands, since when activated they bring about the destruction of the insulin. For similar reasons, the insulin is extracted from the glands with a solvent which electively keeps the enzymes in an inactive state. Various water-miscible organic solvents for insulin can be used for this purpose, such as the lower alcohols and ketones. The preferred solvents are methanol, ethanol, and acetone, and in the United States because of its availability ethanol is employed almost exclusively. The ethanol or similar organic solvent is diluted with water and acidied to prepare the extracting solvent. It has been found that in order to keep the enzymes inactive and insolubilized it is preferable to have the extracting solvent contain from about 50 to 85% organic solvent, and to be at a pI-I below 4.

To prevent any possibility of confusion, it is desired to point out that the tering, enzymes and proteolytic enzymes" are used in the following specfication and claims to refer to both the pro-enzymes and the active enzymes. However, wherever the active or inactive form of the enzymes is important, more specic terminology is employed.

For many years the pancreas gland residues resulting from the extraction of insulin from pancreas glands were discarded. No attempt was made to recover the enzymes from these residues,

6 Claims. (Cl. 195-66) and instead fresh pancreas glands were extracted to obtain the enzymes. This may have been due to a belief that the enzymes were destroyed by the high concentrations of organic solvents used in the insulin extraction. However, it is now known that the enzymes are not destroyed but only precipitated or denatured; and that they can be convertedl back to their normal state by reducing the concentration of the organic solvent to a suiciently loi-.v value, that is, by reducing the volumetric ratio of solvent to water. With the commonly used organid solvents (ethanol, methanol, and acetone), it has been believed to oe sufficient to reduce the concentration of the organic solvent to below 26% by volume.

To facilitate the extraction of the insulin it is customary to comminute or hash the raw pancreas glands to pieces about one-fourth to onehali inch in diameter, and then to contact the comminuted gland tissue with the extracting solvent. The water-organic solvent mixture is generally acidied with a suitable acid, such as sulfuric, hydrochloric, phosphoric, oxalic, etc. After the contacting of the solvent with the gland material for a sufcient period to extract the insulin, the supernatant is separated from the residue, generally by centrifugation. The residue thus obtained generally contains from about 30 to 40% solids, or in other words 60 to 79% of the residue is the extracting solvent containing the high concentration oi organic solvent.

It may contain as little as 50% liquid.

The extraction of the pro-enzymes from the pancreas gland residues can be carried out in a number of diilerent ways. One method which has been employed is to reduce the concentration of the organic solvent in the, residues to below 20% by adding water to the residues to dilute the organic solvent therein. The solids of the residues are then kept in contact with the water containing less than 26% of the organic solvent for at least from 24 to it hours so that the water can extract the enzymes. Depending somewhat on the character of the particular pancreas tissues, the amount of agitation, and' other factors, it is only possible to extract from to 80% o1 the enzymes in a 48 hour period by this method.

In large scale commercial operation extraction periods of 48 hours are undesirable, because the use of the extracting equipment is seriously limited. On the other hand, it is undesirable to discard the residues while they still contain substantial quantities of the enzymes. Therefore, it is desired to develop a method for carrying the extraction of the residues to substantial completion within a greatly reduced period of time.

It is now believed that one reason for the difficulty of extracting the enzymes with Water is that the pancreas glands contain substantial amounts of fat or lipoidal matter which interferes with the extraction. The fat content of pancreas glands varies from about 5 to ci0% by weight. Beef pancreas glands are Irelatively low in fat and contain on the average about 5to 10% fat, While pork pancreas glands are relativelyA high in fat, containing on the average 30 to 46% fat. The iat is distributed in particles throughout the tissue, and it is believed that these particles of fat block the penetration of the Water and shield the enzyme-bearing tissue. This is true because the lipoidal matter is not Water-soluble, and is substantially impermeable to Water. Because of the very high iat content in pork pancreas glands, the interference of the fat therein with the extraction of the enzymes is a partcularly acute problem in extracting enzymes from pork pancreas glands. in the extraction of insulin from comminuted pancreas glands the presence or the fat particles in the pieces of glandular material has not been a serious problem insoar as tending to prevent complete extraction of the insulin. This is probably the case because high concentrations of organic solvents, such as ethanol, methanol, and acetone, are employed. rThe fat or lipoidal matter is quite soluble in such organic solvents and these solvents can penetrate the tissue much more easily than water.

It has been suggested that effective contact between the Water and the pro-enZyme-bearing tissue can be promoted by greatly increasing the degree of subdivisions of the glandular material, for example, by passing the residue through a high speed grinder. This approach, however, has a number of practical limitations and has not proven satisfactory, especially when it is desired to obtain the enzymes in crystalline form. in general, increasing the degree of subdivisions tends to accentuate the dinicuity of obtaining a clean separation between the liquid and solids. Separation of fat particles dispersed in the extract is particularly troublesome, since the fat particles are only of a slightly lower specific gravity than the water. Therefore, on centrifugation the fat particles follow the liquid portion and tend to remain dispersed therein. The presence of such fat particles or other solid particles of the glandular material interferes with the recovery of the pro-enzymes from the extract. This is particularly serious when it is desired to use salting out procedures to recover the enzymes in crystalline form. Therefore, it is advantageous to prepare an extract which is substantially free ol solid particles.

t is therefore an object of this invention to provide a method for carrying the extraction of enzymes from pancreas gland residues to substantial completion Within a greatly reduced period of time, 1t is a further object of this invention to develop an extraction method in which a more effective contact is achieved between the water and the enzyme-bearing tissue Without unduly increasing the subdivision of the glandular material. More specincally, it is an object of this invention to provide a method for removing the fat particles from the pieces of glandular material, and particularly from pork pancreas material, thereby increasing the amount of enzyme-bearing tissue eiectively contacted by the Water. It is a further object of this invention to provide a method in which the Water is forced against and into the exposed tissue during and after the separation of the fat particles therefrom. It is a still 'further object oi this invention to provide a method oi' removing the fat particles from the extract subsequent to their separation from the pieces of glandular material to prevent the interference o the fat particles with the subsequent recovery and puriication of the enzymes. Further objects and advantages Will appear as the specification proceeds.

During the experimental worn leading to this invention, it was discovered that the fat particles could be separated from the pieces ci pancreas gland residue, and the complete extraction of the enzyme achieved, even from pork pancreas residues, within a greatly reduced period oi time by forming an aqueous slurry of the pancreas residue and passing the slurry through a zone Within which the pieces of glandular material are distorted into a succession of irregular shapes. Preferably, the slurry is maintained in a state of violent agitation While simultaneously disto"L ig the pieces of glandular material in the manner described to force the Water into contact with the tissue. It has been found that by this step the fat particles in the pieces of glandular inaterial are loosened and caused to separate from the enzyme-bearing tissue, -which 'thereby increases the amount of the enzyme-bearing tissue effectively contacted by the water. Also, the agitation or churning of the slurry serves to promote the coalescence of the separated rat particles into aggregates, which can be easily separated from the liquid portion. Thus, an extract can be 0btained which is particularly suited for crystallization.

The starting material 'for carrying out the new extraction method of this invention is preferably the residues resulting from the extraction of insulin from comminuted pancreas glands by contacting the glands with a Water-misible organic solvent for insulin. The character oi these residues has already been described in detail. Starting with these residues the rst step in the process of this invention is to form a slurry containing the solids of the residues and a liquid-extracting solvent at least Water by volume. Since the liquid portion of the residues immediately after the extraction of the insulin may have a concentration of above 50% organic solvent, it will generally be necessary to reduce the concentration of the organic solvent in the residues in order to form a slurry for use in this process. More specifically, the slurry for use in the process oi this invention should contain at least 3 parts by weight of an aqueous extracting solvent (containing at least 80% water) to each part of solids, and up to about 30 parts of solvent per part of solids can be used. Preferably, between about to l2 parts of extracting solvent to each part oi solids is employed, and optimirn results appear to be achieved when about 9 parts of extracting solvent are used for each part of solids. it will be understood that when the slurry is formed directly from the ordinary pancreas gland residues resulting from the insulin extraction process that the amount of water to be added can be gauged in terms or" the total Weight of the residues, since the ratio of liquid to solids in the residues ordinarily will be approximately constant.

The particular method of reducing' the concentration'of the organic solvent in the residues and forming a slurry having the desired amount or 5. water therein is not of critical importance for this invention. If desired, the commonly employed method of diluting the residues by adding water thereto until the concentration of organic solvent is reduced to less than 2G% of the liquid portion can be used to form the slurry. For example, if the ratio of liquids to solids is 2 to 1 and the liquid portion of the residues contains 50% organic solvent, one part of water to each part of total residues or 3 parts of water to each part of solids in the residues will be required to reduce the concentration of the solid to around Since it is customary to use around 65% concentrations of the organi-c solvent for the insulin extraction, it will be apparent that it will be necesary to add a greater amount of water to most residues. Also, it has been discovered that the extraction is facilitated if the concentration of the organic solvent in the liquid portion is reduced to at least 7% by volume, and preferably to below 4%. To produce a slurry having a concentration of 4% organic solvent in the liquid from residues having a 65% concentration of organic solvent in the liquid, it would generally be necessary to add somewhat over 30 parts of solvent per part of solids. This would result in a slurry, which although usable in the process of this invention, would have an excessively large volume and would therefore increase the difficulty and expense of carrying out the process.

Various procedures can be employed in addition to the dilution method to produce a slurry of limited total volume with the desired concentration of water in the liquid portion. One method is to rst press the pancreas gland residues to decrease the percentage of liquid therein. Another method is to add the required volume of water to produce the correct concentration, and then to decrease the total volume by centrifuging or decanting. However, it is preferred that the method described in co-pending application United States Serial No. 259,091, led November 30, 1951, be employed. This method is described in detail in the cited application and reference is hereby made to this description. Briefly, the preferred method of forming the slurry is to add from 6 to 14 parts of water to each part of residue solids. The mixture is then disstilled under reduced pressure to decrease the concentration of the organic solvent therein, to at least 20%, and preferably to at least 4% by volurne. During this distillation step the pH is pref erably maintained below pH 4.

The slurry resulting from this procedure is well adapted for use in the process of this invention. Because of the reduced pressure during the distillation the organic solvent can be removed from the mixture while leaving -rnost of the water therein. Ordinarily, it is only necessary to remove about 2 parts of water with each part of organic solvent. For example, when from 6 to 14 parts of water has been added to each part of solids, the slurry subsequent to the distillation step may contain approximately 4 to 12 parts of liquid to each part of solids, and the liquid portion will of course contain not over 20% of the organic solvent. Furthermore, when the concentration of the organic solvent has been reduced to 4%, the aqueous extracting solvent will then contain 96% water.

During the distillation step, depending somewhat on the total time of distillation and the temperature used, a considerable portion of the pro-enzymes will be extracted. Normally this will be from 60 to 80% of the pro-enzymes. Even though the pro-enzymes are more easily extract-V able at the temperatures employed in the distillation step, it is not possible to carry the extraction above about because the required time and temperature will cause the destruction of substantial quantities of the enzymes. Therefore, it is desirable to employ additional procedures for carrying the extraction to substantial completion in a minimum of time while preventing the enzymes from being destroyed. The present invention admirably lls this need. However, it will be understood that the process steps of this invention are not limited to use with a slurry obtained by distillation procedures, and that, if desired, the slurry can be directly formed from the pancreas gland residues and subjected to the process steps which will now be described.

After the formation of the slurry by any of the methods. described above, the slurry is then passed through a zone in which the pieces of glandular material therein are rapidly distorted into a succession of irregular shapes by the action of mechanical forces. The desired distortion can be described as a spreading-distortion which can be defined as any distortion which temporarily enlarges the surface area of a piece of glandular material by twisting, stretching, or mashing. While it is possible to subject the pieces of glandular material to spreading-distortion in the presence of the solvent with a minimum of agitation of the solvent, it is preferred to maintain the slurry in a state of violent agitation while the pieces are being subjected to spreading-distortion to force the solvent against and into the pieces of material. More specilically, it is preferred to impart a rotary churning motion to the slurry while the solids therein are subjected to spreading-distortion. ft is believed that one result obtained, which is the loosening and separation of the fat particles from the glandular material, is due to the cooperation between the water and the action cf the mechanical forces. Also, the rotary churning motion is believed to promote the coalescence of the separated fat particles into larger aggregates, and to prevent the fat particles from becoming dispersed in the liquid portion. The particular mechanical equipment which can be used for carrying out this step will subsequently be discussed in detail.

To prevent destruction of the enzymes, it is desired to maintain the pH of the slurry on the acid side (below pH 6.5) during the treating of the slurry as just described. Preferably, the pH of the slurry is maintained below pH 4 by means of the acids employed in the insulin extraction, since considerable quantities of acid will be presentl in the residues. For example, sulfuric, hydrochloric, phosphoric, oxalic, etc. or mixtures thereof can be employed. The presence of sulphuric acid in the slurry has been found to give particularly good results during the extraction and to facilitate subsequent filtration of the extract.

If the pH of the slurry is maintained below about pH 4, the temperature of the slurry is not especially critical to prevent destruction of the enzymes. In fact, room temperature (around 25 C.) is satisfactory. However, it has been found that lower temperatures are advantageous in promoting the separation of the fat particles from the glandular material and in causing them to coalesce after their separation. In general, temperatures below about 15C. are

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helpful in accomplishing these. results.. When the residues are hog' pancreas gland residues', however, it is advantageous to use. temperatures below C; down to aboutO" C.

After the slurry has been'. treated in the manner described, it is preferred to holdv the slurry in a poolin a substantiallyl stationary condition to permitthe separated iat. particles to continue to coalesce and to rise to the surface-of thepool. The solid pieces or glandular material have a slightly higher specific gravity than that of the aqueous extracting solvent, and therefore will tend to slowly settle to the bottom or" the pool. On the other hand,the fat particles have a slightly lower specific gravity than the aqueous solvent, and therefore will tend to rise to the surface of the pool. Thefatparticles also tend to coalesce to form larger aggregates, and` the coarser aggregates of fat particles can then be removed from the surface oi; the pool by. skimming. While violent stirring or agitation of the slurry been found advantageous to employ agentle-agitation which is `lust sufficient to keep the piecesof glanzlular material in suspension, and thereby prevent the packing of' the: material in the bottom of the pool. Also, the use of gentle agitation appears to improve the rate of separationv of fat particles and to accentuate their tendency to coalesce. Temperatures below C. and preferably below 10 C. are desirable. As indicated above, when the residues are pork pancreas residues temperatures below 5 C1 to. about 0 C. are advantageous because of the larger percentage of fat present in theresidues.

After the step of holding the slurry in the pool to bring about the furthercoalescence of the fat particles and to permit the removal. of" atk least the. coarser aggregates of fat particles by skimming the surface of the pool, it is generally desirable to again pass the slurry through a zone in which the slurry is maintained in. a state ofviolent agitation whilethe pieces of glandular material are distorted into a succession of irregular. shapes..

lI-lowever, this step is not essential, since substantially the same. results can be achieved by increasing the number of passes of the slurry through the zone prior to the holding.' of the slurry in the pool. Best results, however, appear to be achieved by usingv the sequence of steps described in that the total timeY requiredA can be substantially decreased.

At this point in the process, the slurry: will be composed of a liquid portion and: solid pieces of glandular material. The enzymes will bedissolved, mostly inthe form of pro-enzymes, in-the liquid portion, and the remaining fat particles will be suspended in the liquid portion together with the solid pieces of glandular material. The.

ext operations involve the separation of both the solid pieces oi glandular material and the rest of the iat particles from the extract. To

accomplish this, the slurry is first centrifuged to.

separate the solid pieces of the glandular material from the extract. Because the fat parti-Y cles are somewhat lighter than the solvent, they will be contained in the extract. The centrifuging operation has been found effective in still further increasing the extent of coalescence of the fat particles to larger aggregates. The

coalesced particles of fat have a correspondingly greater tendency to float in the extract'. Therefore, followingcentrifugation the aggregates can.

easily be separated from the. extract. One satisfactory method is.- to. hold; the'. extract in` a: pool.

while it is in the'poolis undesirable, it has long enough to allow the' aggregates to oat tol the surface, and then to remove ming..

The extract will then be substantially free of a-tparticles which permits it to be subjected to further process steps to recover the enzymes without interference from the fat particles. As indicated above, this. is particularly important when it is desired to recover the enzymes in crystalline-A form. The failure of prior workers to prepare crystalline enzymes from pork pancreas is probably due. tothe fact that they did not know how to prepare a fat-free extract. Therefore, itis believed to be a signicant advantage of the method.4 of this invention that it for the rst time provides a simple and convenient method of preparing crystalline enzymes from pork pancreas residues.

The fat-free extract will contain the enzymes mostly inthe form of. their precursors-which are termed the pro-enzymes, as explained above. The pro-enzymes canbe separated and activated bywell. known procedures. The various enzymes and. inert protein impurities have diiferential them by skimsolub'ilitiesin` aqueous ammonium sulphate solutions, and. therefore ammonium sulphate is generally employed to saltV out the impurities and the enzyrnesin the order desired. After the enzymes are salted out, they can be further purified by recrystallization or other purification 'ethods to produce as products the crystalline enzymes. of standard potency and stabilized strength.

To facilitate the understanding of the process v steps described above and the means of carrying out these steps, Eig. l of the accompanying drawing shows in. diagrammatic form a simplified flow sheetv for. removing the enzymes from pancreas residues resultingfrorn the extraction of insulin from pancreas glands. In the upper left-hand corner therev is shown a storage tank lil into which the pancreas residues andthe required amount of acidied. Water' are charged. This. mixture is pumpedfromtank it into steam still H. in. which the organic solvent is removed byV distillation. under reduced pressure. The organic solvent, which; will generally be ethyl alcohol, leaves through. the top of steam still H. and is passed through a condenser i2 in which it is condensed'into a liquidand passed into a tank lt.

The slurry'remaining in steam still I i can then be. passedtot an extractor it. In the illustration given, extractor i@ isv equipped with. a jacket through. which. cold brine can be circulated to refrigerate. the slurry. Extractor lil is also equipped. with an agitator i5. It is preferred to hold. the slurry in. extractor i4 long enough to permit it to be cooledl to at leastv 15 C., during which time agitator t5 can be operated to keep the solid pieces of theslurry in suspension. After the slurry is cooled to the desired temperature it is then passedby means of outletline i5 in the bottom of the' extractor through pump. il and backk into the. top or the extractor by means of line i8.

Within pump i' the. slurry is maintained in a state of-.violent agitationwhile simultaneously distorting the pieces of glandular material intol a succession of irregular shapes. It has been found that an impeller-type pump is the most suitable apparatusior accomplishing this result. A. perspective view of pump Il1v with the parts in separated. relation is shown in Fig. 2. In the illustration given, pump ll comprises a cylindricalcasin'gi Ha. adapted to house a heavyebladed accanita Separation was accomplished in a Bird centrifuge or the cylindrical type. The tissue from the iirst centrifugation was suspended in 225 gals. o tap water containing 340 c. c. of H2304. The washing was accomplished by a Sil-minute circulation through the impeller-type pump. The resultant suspension was then centrifuged and the efliuent added to that obtained from the irst separation.

0.7 lb. of (Nl-102504 was added per gallon of extract. This afforded a .15 saturation with the salt. The salt was added slowly in order to prevent a localized high concentration and a poten tial irreversible precipitation of protein. 45 minutes of inipeller-type agitation was found to be suiicient for the complete dissolution of the salt.

The separation was accomplished in a Sperrytype press which had been pre-coated with Hiflo. The pl-l at this point was found to be critical (1.9-2.1 before salt addition) for there are semisoluble materials present which. if not precipitated at this pH, not only clog the filter press, but also pass through in the eluent and interfere with subsequent processing. The press was washed with 50 gals. of water containing 35 lbs. of (NHilQSOi, 75 c. c. of H2804 and 5 lbs. of I-liflo to which had been added the iat removed during the extraction and centrifugation steps. rfhis washing was followed by another il-gal. portion of the previous washing solution. Air was blown through the press for four hours in order to complete recovery of the filtrate.

4 lbs. of (NHDZSCM was added per gallon of filtrate obtained. The solution was then ap proximately 0.8 sat. with (NH4) S04. Dissolution was completed by a 12S-minute circulation through the impeller-type pump and precipitation was obtained through a Li-hour agitation with an impeller-type stirrer. This suspension was then filtered through a Sperry-type press, pre-coated with l-lilo. iflo was also added to the slurry; the total amount used being 12 lbs. The nltrate was a protein-free (NH4) 2SO4 solution, which can be recovered. The filter cake was dissolved in 5 c. c. of distilled water per gram of cake. The pH was held at 2.8-3.3. 150 g. (NHrlzSOi was added per liter of solution. The addition was made slowly and the material was agitated vigorously for 1.5 minutes and allowed to settle for an additional 30 minutes before starting the ltration. YThe press was washed with liters of a 0.4 saturated (Nl-14) zSOr solution. The preipitate was held at 23 F. for further processing.

ZGG g. of (N1-192504 was added per liter of filtrate obtained from the last step. The suspension was agitated for 15 minutes and allowed to nocculate over an additional SG-minute period. The material was then iiltered and the precipitate removed to a temperature of 65 F. for further processing. This precipitate contained the crude trypsinogen and chymotrypsinogen.

110 g. ci (Nl-14h50.. per liter of solution was added to the nitrate from the last step and the ltered precipitate obtained therefrom was held at 23 F. for further processing. The precipitate Was the crude ribonuclease. The precipitate of crude trypsinogen and chymotrypsin-ogen was dissolved in 1.5 oc. of distilled water per gram of cake. 0.5 c c. of saturated (NllilzSO:L solution was added per gram of cake, slowly, while the material was agitated. The pH oi the solution was then adjusted to 5.0 with 5 N NaOH@ The solution was seeded with chymctrypsinogen crystals and held 16 hrs. at 65 F. for crystallization oi the chymctrypsinogen. Separation was accomplished by vacuum filtration, using Hiflc as a filter aid. The chymotrypsinogen crystals were held at 23 F. for further processing.

To the filtrate from the last step was added: (l) 1.5 cc. of distilled water per grani of cake (2) 1.5 cc. of saturated (NHQESOi solution per gram of cake. After 15 minutes of agitation and 3i) minutes of settling, the suspension was filtered with Hiflo and the precipitate discarded. g. of (Nl-192504 was added per liter of nitrate obtained. After the previously described agitation and settling periods, the material was l tered. The filter calce containing the trypsinogen was dried completely as possible in preparation for crystallization. The trypsincgen cake was cooled to 45 by allowing it to stand in a metal pot placed in a freezer vault. Borate buicr (pH 9.0) was cooled to 60 F. by standing in the cold. The buier solution was then mixed into the trypsinogen cake very slowly in the ratio of 1.5 cc. pe gram of cake with minimal agitation at 32 F. The temperature should never rise about 511 F. This material was allowed tc stand 16 hrs. at 32 F. at pH '7.6. One cc. or saturated MgSOf. solution per cc. of buffer* cake solution. was added slowly, with continuous agitation. This solution was allowed to stand 3--4 hrs. at 32 F. The solution was seeded with crystalline trypsin and held at 5 C. for L.iS-'I2 hrs. for crystallization. The crystalline trypsin was then separated by iiltration. Subsequently, the berate buffer salt was removed by dialysis. and after clarification the dialyzed material was packaged in vials.

Example II rlhe procedure of Example I' was substantially followed except that pork pancreas residues were substituted for the beef pancreas residues. Because ci' the greaterv fat or lipoidal pancreas glands (averaging 30 to 40% as compared to 5 to' 10% for beef pancreas glands) special precautions were taken to prevent the :Eat from interfering with the recovery of the crystalline enzymes. Subsequent to the distillation step, the slurry was refrigerated to around 31 F., and maintained at this temperature during the pump eX- traction and centrifugation steps to promete clotting and separation of the fat particles.

Another modioation in the procedure was the passing of the centrifugate into a small tank immediately after the separation ci the spent residues. In this skimmer tank, the centrifugato was held up long enough to allow the fat particles, which had been further coalesced by the centrifugation, to rise to the surface and to be removed by skimming. In this way, a substantially fat-free extract was obtained.

Example III Crystalline trypsin and chymotrypsin can he recovered from fresh pancreas glands by the following procedure: Ground pancreas glands are suspended in 2 parts by weight of water acidied with sulfuric acid to .25 N. The resulting slurry will generally contain approximately 11 parts by weight of liquid to eachpart by Weight of solids. The slurry is then passed to a jaclieted tank in which it is cooled to about 40 F. for beef pan creas glands and to about 31 FL for pork pancreas glands. The slurry is then circulated from the tank through an impeller-type pump and back into the tank for a period of 15 minutes. The pump is stopped and the slurry is gently agitated forv 15 minutes, and the coarser aggregates of fat are simultaneously removed by skimming. The pump is started again and the slurry is passed through the pump for another 15 minute circulation cycle.

The solid glandular material is then separated by centrifugation, and held for the extraction of the insulin therefrom. The centrifugation operation will have caused the separated fat particles to further coalesce, and they can then be removed by the procedure described in Ex ample Il. rThe removal of the rest of the fat from the extract at this point is particularly iinportant when the starting material is hog panl creas glands.

To the centrifugate is added ammonium sulfate until a concentration of 0.4 saturation is reached. The resultant suspension is allowed to settle at C. for 48 hours.

The suspension is then ltered and the pren cipitate, containing the animal protein fractionation, is discarded. Ammonium sulfate is added to the supernatant to 0.7 saturation, and the resultant suspension is allowed to settle for 48 hours at 5 C. The crude enzyme precipitate is separated, and the supernatant discarded.

The crude 0.7 saturated cake is reworked by dissolving it in volumes of distilled water and refractionating it through the 0.4 and 0.7 saturated ammonium suliate steps at 5 C., as described above. The 0.7 saturated Cake obtained by these steps can be reworked again by dissolving it in 3 volumes of distilled water and reiractionating through the 0.4 and 0.7 ammonium sulfate saturation steps at 25 C.

The 0.7 saturated cake thus obtained is dissolved in 1.5 volumes of distilled water at 25 C. Saturated ammonium sulfate solution is added to 0.25 saturation. The pH is adjusted to 5.0 with 5 NaOH. The solution is inoculated with a small amount of chymotrypsinogen crystals, and allowed to stand at 25 C. until the chymotrypsinogen crystallization is complete, usually 48 to 72 hours will be required. The suspension is then filtered and the precipitate held for crystalline chymotrypsin preparation.

The pil of the ltrate subsequent to the separation of the crude chymotrypsinogen is adjusted to 3.0 with 5N H2504. Ammonium sulfate is added to this solution to 0.7 saturation. The suspension is iiltered and the supernatant discarded. The precipitate obtained is dissolved in 3 volumes of distilled water and reworked through a 0.4 and 0.7 ammonium sulfate saturation at 25 C. The 0.7 saturated precipitate is dissolved in 1.5 volurnes of pH 9.0 borate buer at 10 C. The pH of the solution is adjusted to 7.0 with 5N NaCl-I. Saturated magnesium sulfate is added to 0.5 saturation. The solution is held at 5 C. until crystallization or" the trypsin is complete, usually 7 to 9 days will be required. The crystallization suspension is separated by filtration and the ltrate discarded. The crystalline trypsin cake may then be held at 5 C. or suspended in 10 volumes of. water and lyophalized.

The precipitate of crude crystalline chyn1otrypsinogen is reworked by suspending it in 3 volumes of distilled water. Saturated ammonium sulfate solution is added to 0.5 saturation. The precipitate obtained is separated and can be again reworked by dissolving it in 3 volumes of distilled water and adding the ammonium sulfate solution to 0.5 saturation. The twice crystallized cake is then suspended in 3 volumes of distilled water and 1 volume of pl-I 7.5 M sulfate buffer is then added. The pH of the solution is adjusted to 7.6,

and the solution is seated with crystalline trypsin. Activation occurs at 5 C. after a period of 48 hours. The pH of the activated solution is then adjusted to 4.0, and the activated material is precipitated by adding ammonium sulfate to 0.5 saturation. The 0.5 precipitate is separated, and dissolved in 3A volume of acidiled water with the pH adjusted to 3.9, and crystallized at 25 C., usually 24 to 48 hours will be required to complete the crystallization. The crystallized chymotrypsin can then be separated and recrystallized by dissolving in 1.5 volumes of pH 2.0 water. Saturated ammonium sulfate is added to 0.5 saturation, and the suspension held for a few hours at 25 C. `rIhe resulting precipitate of crystalline chymotrypsin is recovered by iiltration, the lter cake washed with saturated magnesium sulfate solution at pH 1.3, and held at 5 C.

Example I V Crystalline trypsin can be prepared by the iol lowing procedure: 7 parts oi water are added to each part of pancreas gland residues subsequent to the insulin extraction to form a slurry con taining at least 2l parts of liquid to each part or" solids. The slurry is then passed into a tank in which it is cooled to at least 40 F. After reaching the desired temperature, the slurry is circulated through a 3 bladed impeller-type pump for 15 minutes, allowed to stand with slight agitation in the tank for 15 minutes, and again circulated through the pump for 15 minutes. A portion of the separated fat can be removed while the slurry is standing in the tank between the circulation cycles by skimming.

rThe extract is separated from the spent residues by centrifugation, clarified, and a crude trypsinogcn precipitate is obtained at 0.8 saturation with ammonium sulfate. This precipitate is separated by iiltration and the iilter cake is dissolved in 6 volumes of water, and purication is effected by adding a saturated ammonium sulfate solution at a concentration between 0.35 to 0.55 saturation. The trypsin is then crystallized at pH 7.1 to 7.5 in a berate buffer solution at 0.3 saturation with magnesium sulfate. The berate salt is removed from the crystalline trypsin by dialysis, and after clariiication the ialyzed material is packaged in vials.

Eample V For rapid handling of the residues to obtain crystalline trypsin and chymotrypsin the following procedure can be used: Suspend 1,000 lbs. of residue in 400 gals. of water containing 2200 cc. sulfuric acid. Remove gals. of liquid by distillation under reduced pressure. Cool the concentrate to 32 F. Circulate through an impellertype pump| for 15 minutes. Centrifuge and skim the fat from the eiiluent in a skimmer tank. Suspend the residue in 200 gals. of water containing 300 cc. sulfuric acid. Circulate through an impeller-type pump for 15 minutes. Centrifuge and skim the eiiiuent as before. Combine the` extracts and precipitate the trypsinogen and chymotrypsinogen between 0.4 and 0.7 saturation with ammonium sulfate, after first precipitating and separating the inert contaminants at 0.4 ammonium sulfate saturation. The crude enzyme cake thus obtained can be reworked to obtain the trypsin and chymotrypsin in crystalline form by the procedure set out in Example III.

While in the foregoing specification this invention has been described in considerable detail for purpose of illustrating embodiments lthereof, it will be apparent that many of these details can be varied widely Without departing from creas glands by contacting said comniinutedr glands with a water-miscible organic solvent for insulin, the steps of forming a slurry containing the solid pieces of the glandular material in said residues in admixture with from 6 to 12 parts by Weight of an aqueous extracting solvent to each part of said material, said extracting solvent containing at least 96% Water, cooling said slurry to a temperature between about 0 C. to 15 C., and passing said slurry a plurality of times through a zone within which said slurry is maintained ina state of violent agitation while simul taneously distorting said pieces of glandular material into a succession of irregular shapes, Where by the fat particles in said pieces of glandular niaterial are loosened and caused to separate from the enzyme-bearing tissues, thereby increasing the ain of said enzyme-bearing tissues effectively contacted by the water With the result that said pro-enzymes can be completely extracted in a greatly reduced period of time.

2. In a process for extracting trypsin and other o proteolytic enzymes from residues resulting from the extraction of insulin from comminuted pancreas glands by contacting said coznrninuted glands with a water-miscible organic solvent for insulin, said residues being in -the ferm of solid pieces of glandular material having particles of fat embedded therein, the steps of forming a slurry containing the solid pieces of glandular material from said residues in admixture with an aqueous extracting solvent containing at least 80% Water, and passing said slurry at a tempera- .ture between 15 C. through a zone within which said pieces of glandular material are subjected to spreading-distortion While a rotary churning motion is simultaneously imparted to said slurry, thereby simultaneously extracting said trypsin and other enzymes and liberating and coalescing said iat particles.

3. The steps of claim 2 wherein the residues are hog pancreas gland residues.

4. In a process for preparing an enzyme extract from which crystalline trypsin can be obtained wherein the starting material consists of insulin-free pancreas gland residues composed oi' solid pieces or enzyrneebearing glandular material having particles of fat embedded therein, said pieces ci glandular material being suspended in a mixture of Water and from 50 to 85% by volume of an organic solvent selected from the group consisting of methanol, ethanol, and acetone, the steps of forming a slurry containing the solid pieces of glandular material from said residues in admixture with an aqueous extracting solvent containing at least 80% water and .being at a pl-I below 4, and passing said slurry at a temperature below 15 C. through a zone within which said pieces of glandular material are distorted and forced into contact with said aqueous extracting solvent While a rotary churning motion is simultaneously imparted. to said slurry, thereby simultaneously extracting trypsin and liberating noalescing said iat particles.

5. .in a process for obtaining crystalline trypsin and other proteolytic enzymes from pancreas gland residue solids resulting from the extraction of insulin from comminuted pancreas glands by contacting said cornrninuted glande with a ivaternniscible organic solvent for insulin, said residue solids containing particles of fat enibedded therein, the steps of forming a slurry containing said residue solids in adrnixture with an aqueous extracting solvent containing a sufficient proportion o Water to solubilize the pro- 1 teolytic enzymes in said residue solids, and then pumping said slurry through an inipeller type pump by means of said puinp to produce a rapid extraction of proteolytic enzymes into said aqueous extracting solvent While at the saine time liberating and coalescng said particles of fat, said pumping being carried out with said slurry at a temperature below 15 C. and above the freezing point thereof.

6. In a process for preparing an enzyme extract from which crystalline trypsin can be obtained, the steps of forming a slurry from pancreas gland residue solids, said residue solids resulting from the extraction ef insulin from pancreas glands, said slurry containing suflicient Water to solubiliae the trypsin in said residue solids, and passing said slurry at a temperature not substantially above 5 C. through a zone within which said residue solids are distorted and forced into contact with the water in said slurry.

References Cited in the le of this patent UNITED STATES PATENTS Number Name Date 412,836 Carnrici: Oct. 15, 1889 2,524,858 Fredericksen Oct. 3, 1950 2,529,152 Lindsay Nov. '7, 1950 2,571,126 Frederickson Oct. 16, 1951 FOREIGN PATENTS Number Country Date 613,030 Great Britain of 1945 618,174 Great Britain of 1946 

1. IN A PROCESS FOR EXTRACTING TRYPSIN AND OTHER PROTEOLYTIC ENZYMES FROM RESIDUES RESULTED PANTHE EXTRACTION OF INSULIN FROM COMMINUTED PANCREAS GLANDS BY CONTACTING SAID COMMINUTED GLANDS WITH A WATER-MISCIBLE ORGANIC SOLVENT FOR INSULIN, THE STEPS OF FORMING A SLURRY CONTAINING THE SOLID PIECES OF THE GLANDULAR MATERIAL IN SAID RESIDUES IN ADMIXTURE WITH FROM 6 TO 12 PARTS BY WEIGHT OF AN AQUEOUS EXTRACTING SOLVENT TO EACH PART OF SAID MATERIAL, SAID EXTRACTING SOLVENT CONTAINING AT LEAST 96% WATER, COOLING SAID SLURRY TO A TEMPERATURE BETWEEN ABOUT 0* C. TO 15* C., AND PASSING SAID SLURRY A PLURALITY OF TIMES THROUGH A ZONE WITHIN WHICH SAID SLURRY IS MAINTAINED IN A STATE OF VIOLENT AGITATION WHILE SIMULTANEOUSLY DISTORTING SAID PIECES OF GLANDULAR MATERIAL INTO A SUCCESSION OF IRRUGLAR SHAPES, WHEREBY THE FAT PARTICLES IN SAID PIECES OF GLANDULAR MATERIAL ARE LOOSENED AND CAUSED TO SEPARATED FROM THE ENZYME-BEARING TISSUES, THEREBY INCREASING THE AMOUNT OF SAID ENZYME-BEARING TISSUES EFFEC- 